Parasitology Basic Identification methods by Dr.T.V.Rao MD

1. HUMAN PARASITOLOGY
BASIC IDENTIFICATION METHODS
Dr.T.V.Rao MD
18-01-2018 Dr.T.V.Rao MD 1

2. BASIC TERMINOLOGY AND PRINCIPLES
• Symbiosis: Living together
• Commensalism: One symbiont benefits,
other unaffected
• Mutualism: Both symbionts benefit
• Parasitism: One symbiont
benefits, other is damaged
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3. The Reality of Parasites
• 1.3 billion persons infected with
Ascaris (1: 4 persons on earth)
• 300 million with
Schistosomiasis
• 100 million new malaria cases/
year
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4. 4
Laboratory Methods For Parasites In Faeces
• No technique is 100% successful in detecting
parasites by a single stool examination, and at least
three serial stools must be examined before a
patient can be considered free from infections in
which stages of parasites would be expected to be
found in the faeces.
• Whilst clinical symptoms or a case history may
provide clues as to which parasites may be present,
each faecal specimen should be treated as an
unknown, as parasite stages unrelated to the clinical
picture may be present.
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5. 5
Faecal specimens may contain several
stages of Parasites
• Faecal specimens are examined for the presence of
protozoa and helminthes larvae or eggs.
• The stages of protozoa found in stools are
trophozoites and cysts. The stages of helminthes
usually found in stools are eggs and larvae, though
whole adult’s worms or segments of worms may also
be seen. Adult worms and segments of tapeworms
are usually visible to the naked eye, but eggs, larvae,
trophozoites, and cysts can be seen only with the
microscope.
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6. 6
Collection of faecal specimens
1. Because of the fragile nature of many intestinal
parasites, and the need to maintain their
morphology for accurate identification, reliable
microscopic diagnosis can’t be made unless the stool
is collected properly.
2. Approximately 10 grams of fresh faeces
uncontaminated by urine, oil, water, dyes or radio-
opaque into a clean plastic container.
3. The container should be free from antiseptics and
disinfectants.
4. Label all samples clearly with the patient’s name,
reference number, date, and time of collection.
18-01-2018 Dr.T.V.Rao MD

7. Collection of faecal specimens
5. All samples should be accompanied by a
requisition form from the physician
giving relevant clinical details and recent
travel history.
6. Samples and forms from patients with a
confirmed or suspected diagnosis of
certain infectious diseases such as AIDS
or hepatitis should be clearly labeled
with “Risk of Infection” or “Biohazard”
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8. Collection of faecal specimens
• 7.Most viable parasites are susceptible to
desiccation or temperature variation. If time
lapse between collection and observation is
considerable, i.e. more than 4 days, it may be
necessary to add some form of preservative to
the faeces to retain the morphology as near to
the original as possible.
• 8. Formed samples can be kept in a refrigerator at
+ 4c for a short while, but not in incubator.
• 9. Any whole worms or segments passed should
be placed in a separate container
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9. Collect the Information of the
Patient
• History (Age, occupation, residency, previous
infection)
• Complaint
• Clinical examination
• Investigations
- Laboratory investigations
- Radiology
- Surgical intervention (Exploratory)
Provisional diagnosis
Confirm the diagnosis
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10. 10
The Microscopy in Parasitology
• The Microscope is the parasitologist’s main
tool. If possible the Microscope- should be
binocular; most suitable objectives are the
x10, x40, and x100.
• The Microscope must be covered and
immersion oil removed from the lens -with
xylene or ether when not in use.
• Calibration of the Microscope Eyepiece
Micrometer:
–On many occasions measuring the size of
suspected parasites in faeces is helpful for
identification.(eyepiece micrometer)18-01-2018 Dr.T.V.Rao MD

11. 11
Microscopic Examination of
Wet Mount
• Wet mount is the simplest and easiest
technique for the examination of faeces, and
this method should be performed in all
laboratories at the peripheral level.
• A wet mount can be prepared directly from
faecal material or from concentrated
specimens. The basic types of wet mount that
should be used for each faecal examination
are saline, iodine, and buffered methylene
blue.
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12. 12
The saline wet mount
• Is used for the initial microscopic
examination of stools. It is employed
primarily to demonstrate worm's eggs,
larvae, protozoan trophozoites, and
cysts.
• This type of mount can also reveal the
presence of red blood cells and white
blood cells.
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13. 13
The Iodine wet mount
• Is used mainly to stain glycogen and the
nuclei of cysts, if present. Cysts can
usually be specifically identified in this
mount.
• The buffered methylene blue (BMB) wet
mount should be prepared each time
amoebic trophozoites are seen in a saline
wet mount, or when their presence is
suspected.18-01-2018 Dr.T.V.Rao MD

14. Direct saline and iodine mounts
1. With a wax
pencil writes
the patient’s
name or
number and
the date at the
left-hand end
of the slide.
1418-01-2018 Dr.T.V.Rao MD

15. Preparing a faecal specimen
• Place a drop of saline in
the centre of the left
half of the slide and
place a drop of iodine
solution in the centre of
the right half of the
slide.
• Note: If the presence of
amoebic trophozoites is
suspected, warm saline
(37c) should be used.
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16. Preparation of Wet film
• With an applicator
stick (match or
tooth pick), pick
up a small portion
of the specimen
(size of a match
head) and mix the
drop of saline.
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17. MACROSCOPIC
EXAMINATION
COLOUR
Pale
CONSISTENCY
-Liquid (Troph)
-Formed (Cyst)
-Semi formed (Cyst)
COMPOSITION
?? Blood ?? Mucus
(dysentry)
Adult PARASITES
*Ascaris worm
*E. vermicularis
*T. saginata
STOOL EXAMINATION
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18. DIAGNOSIS
DIRECT
Urine
Stool
Sputum
Biopsy
Blood
Aspirates
INDIRECT
IHAT
LAT
IFAT
ELISA
MOLECULAR
PCR
DNA probes
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19. URINE
EXAMINATION
MACROSCOPIC
colour
white
Chyluria
Filaria
smoky
Blood
S.
haematobium
MICROSCOPIC
Sedimentation
concentration
Membrane
filtration
Acetic acid
RBC haemolysis
Clear ova
Ether
Dissolve fat
M.f18-01-2018 Dr.T.V.Rao MD 19

20. 20
Examination
1. Put the slide with the mounts on the
microscope stage and focus on the mount
with the x10 or low-power objective.
2. Regulate the light in the microscope field with
the sub stage diaphragm. You should be able
to see objects in the field distinctly. Too much
or too little light is not good.
3. Examine the entire coverslip area with the
x10 objective; focus the objective on the top
left-hand corner and move the slide
systematically backwards and forwards, or up
and down.
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21. 21
Examination Cont.
4. When organisms or suspicious material are
seen, switch to the high-dry objective, and
increase the light by opening the sub stage
diaphragm to observe the detailed
morphology.
-This is a systematic examination. If mounts are
examined in this way, any parasites present
will usually be found. If the mount is not
examined systematically, parasites may be
missed. Examine each microscope field
carefully, focusing up and down, before
moving to the next field.
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22. 2218-01-2018 Dr.T.V.Rao MD

23. STOOL EXAMINATION
A Rapid Methods
Saline smear Iodine smear
saline Iodine 1%
Huge number of:
•Eggs
• Protozoal troph. Motility
(Amoeb, flagellates)
Huge number of:
•Cyst morphological details
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24. Need for Concentration Methods for
Faecal examination
• A concentration
procedure is performed
mainly to separate the
parasites from faecal
debris. The concentration
procedure not only
increases the numbers of
parasites in the sediment
but it also unmasks them,
making them more visible
by removing organic and
inorganic debris
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25. STOOL EXAMINATION
Scanty infection
Concentration techniques
Sedimentation Floatation
• Heavy eggs (Ascaris egg)
• Operculated eggs (Trematodes)
• Larvae (Strong sterc.)
• Cysts
• Non Operculated eggs
Trematodes ( S. m.)
Cestode
Nematode(Hookworms,Trichostong)
• Cysts
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26. STOOL EXAMINATION
Saline sedimentation
10 g stool
Saline
Mesh wire gauze
Conical flask
Sediment
Emulsify
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27. STOOL EXAMINATION
Kato technique
Mesh screen
Template
Hole
Remove the template
Cellophane soaked by glycerin
(clears faeces(
Egg count/ g stool
Egg quant. Of: Ascaris, T. trich., Hookworms, S. mansoni
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28. Stool examination other
Texhniques
• Stoll’s technique for eggs of Ascaris,
T. trichiura., Hookworms, S. mansoni
• Baermann’s technique Detec. Of
Nematode L. /stool, soil
• Cultures for Nematode larvae using Filter
paper culture for Larvae of: St. stercoralis
(A,L) and Hookworms
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29. 29
Artifacts
• Artifacts other things, living or
artificial, present in the stool that are
not parasites and could mislead the
laboratory worker.
• Note: “Artifacts not to be mistaken
for cysts”.
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30. 3018-01-2018 Dr.T.V.Rao MD

31. 31
Plant cells
Air bubble
Plant hairs
Plant fibre
Pollen grains
Non-human
coccidial oocysts Fat droplets
Soapy plaques
Starch cell Charcot leyden crystals Muscle fibers
Fatty acids Macrophage
Epithelial cells18-01-2018 Dr.T.V.Rao MD

32. •Round worm
•Hook worm
•Tape worm
•Pin worm
•Whipworm
PARASITE
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33. MICROSCOPIC
EXAMINATION
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34. PIN WORM EGG COLLECTION
Eggs of Pin worm – Enterobius vermicularis rarely
appear in stools. These are usually collected in
the folds of skin in perianal region.
COLLECTION
Cotton swab / Plaster patch – Anus especially in
early morning – Dipped in Saline – Observed.
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35. EXAMINATION OF PARASITES
Warm stools are best for detecting Ova or
parasites. Do not refrigerate the specimen.
Because of cyclic life cycle of parasites, three
separate random stool specimens are
recommended for examination.
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37. NORMAL VALUES
• Undigested food materials – None to small
amount
• Starch – None
• Eggs, Cysts, Parasitic fragments – None
• Yeasts – None
• Leukocytes – None
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38. HOOKWORM
Ancylostoma duodenale.
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39. TAPEWORM
Taenia solium-Pork
Taenia saginata-Beef
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40. WHIPWORM
Trichuris trichura
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41. PINWORM
Enterobius vermicularis
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42. ENTAMOEBA
Entamoeba histolytica
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43. GIADIASIS
Giardia lamblia
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44. Examining other Specimens
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45. SPUTUM
EXAMINATION
MACROSCOPIC
Appearance
Bloody (Parag)
Rusty brown
(Parag)
MICROSCOPIC
Concentration
NaOH
Sputum
Centrifuge
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46. BIOPSY
SPECIMEN
SKIN
O. Volvulus
mf
MUSCLE
BIOPSY
T. Spiralis
larvae
RECTAL
BIOPSY
Schistosomes
egg
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47. ASPIRATES EXAMINATION
CSF
• Afr. Tryp. (trypom)
• FLA (troph)
• M.f. of loa loa
• L. of T spiralis
Duodenal aspirates
• G. lamb troph
• Crypto oocyst
• St sterc. Rh L.
• Fasciola eggs
BM aspirates
• L. donovani a,ast.
•T. cruzi amast.
•P. falciparum.
Cutanoeus ucler
•Leishmaniasis
Direct stain (amast)
NNN (promast)
•Lumbar puncutre
• Centrifuge
•Examine sed.
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48. BLOOD
EXAMINATION
Blood films
Thin Thick
Buffy coat
films
QBC technique
Knott’s conc.
tech.
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49. BLOOD EXAMINATION
BLOOD FILMS
• Thin • Thick
Bld drop
spread
Air dry
methyl alcohol
Geimsa
Air dry
Geimsa
Circular motion
Malaria, Babesia, Filaria, Tryp.
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50. Observe the Thin and Thick Smear
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51. QBC Method is used in ….
• The QBC Malaria method is the simplest and
most sensitive method for diagnosing the
following diseases.
• Malaria
• Babesiosis
• Trypanosomiasis (Chagas disease, Sleeping
Sickness)
• Filariasis (Elephantiasis, Loa-Loa)
• Relapsing Fever (Borreliosis)
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52. QBC A QUICKER ALTERNATIVE IN
MALARIA
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53. How to read the QBC results ….
• When the operator looks
through the wall of the
tube, the nucleus of the
parasite fluoresces bright
green, and the cytoplasm
shows up as yellow-orange.
The shape and colours are
quite characteristic, and
since the parasites are
concentrated up to 1000X,
there are usually a large
number of them in any field
of view in this area of the
tube.
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54. URINE EXAMINATION
SEDIMENTATION CONCENTRATION
15-20 min Centrifuge (2 min)
Clean conical
glass receptacle
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55. URINE EXAMINATION
Membrane filtration technique
air
10 ml urine
Nucleopore filter
Eggs of Schistosoma
+ Saline
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56. URINE EXAMINATION
HELMINTHES
• S. haem.egg
• E. vermic. egg
• S. mansoni egg
• Mf (Ov, Wb)
• H sand
PROTOZOA
• T. Vag troph
ARTHROPODES
• Pthirus pubis
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57. Indirect immunological diagnosis
• Serology – All tests
available
– IHA
– ELISA
• More useful in
– Amoebiasis
– Leishmaniasis
– Malaria
– Toxoplasmosis
– Trichinosis
– Filariasis
– Echinococcosis
• Skin Tests –
Specificity low,
cross reactions
common
–Casoni’s test
–Leishmanin test
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58. • Program created by Dr.T.V.Rao MD
for Basic Learning methods to
diagnose Parasitic Human Infections
• Email
• doctortvrao@gmail.com
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